Several approaches to improve the efficiency of gene transfer into reconstituting hematopoietic stem cells are presently being evaluated by the Simian Gene Transfer and Bone Marrow Transplantation Program of the National Heart, Lung, and Blood Institute. These involve methods for 1) expansion of populations of cycling progenitors and stem cells in vitro and in vivo using hematopoietic growth factors; 2) isolation of cellular fractions having a high proliferative capacity from bone marrow(BM), peripheral blood(PB), and neonatal cord blood; and 3) improving transduction efficiencies by using different methods for gene transfer. Over the past year, we have used a retroviral vector that uses the gibbon ape leukemia virus(GaLV) envelope protein to determine the viruses tropism, as well as an adeno-associated virus(AAV) vector to transduce immunoselected cells. Transduced cell types have included cytokine mobilized PB CD34+ cells, BM-derived CD34+ cells, and CD4- enriched lymphocytes. Immunoselected BM and PB CD34+ cells were characterized by phenotype and cell cycle status from rhesus macaques and used in autologous BM and PB transplants. AAV and GaLV transduced BM and GaLV transduced PB-derived CD34+ cells were reinfused into animals that had received 6.5GYx2 total body gamma-irradiation (TBI), while GaLV transduced CD4-enriched lymphocytes were reinfused into non- irradiated animals. Circulating leukocytes were analyzed at regular intervals for evidence of gene transfer using a polymerase chain reaction(PCR) assay. Animals transplanted with either the GaLV retroviral vector or with the AAV had 1% or less of their circulating leukocytes marked with the transduced gene. In the case of the GaLV transduced CD4-enriched T-lymphocytes, however,as many as 10% of the circulating leukocytes contained the transduced gene. With continued improvements in vector design, and continued advancements in the understanding of stem cell biology and gene regulation, studies utilizing primate models will prove of major importance in developing suitable protocols permitting safe and effective gene transfer into human subjects.